Human embryonic stem cell (hESC) research has a great potential to further our understanding of basic mechanisms of human development and may lead to the development of novel therapeutics. Absent from this young field has been the acceptance and use of reference standard cell lines. Uniform use of a reference standard would permit investigators to reliably compare results across laboratories and would aid in building sequential advances in the field. A reference cell line should be easy to obtain; the cell line should be relatively easy to grow and maintain; there should be available data for the comparison of results; data and experiments should be easily reproducible; and comparable results using the cell line should be obtained across experiments and across laboratories. In addition to these properties, a reference standard should represent, as closely as possible, the normal or experimental cell line and a particular state of that cell line. In this grant application, we are proposing a reference standard for the undifferentiated hESC state. We propose that nullipotent 2102Ep is better representative of the undifferentiated hESC state than any non-hESC line. Currently, however, there is no commercial source of standardized 2102Ep. We propose to: 1. Perform side-by-side characterization and comparison of two clones of the human embryonal carcinoma cell line 2102Ep 2102Ep cl.4D3 and 2102Ep cl.2A6 - in order to determine which clone is best suited as a reference standard for the undifferentiated hESC state. The criteria used to determine which clone is best will be based on elevated, uniform expression of markers of the undifferentiated state. 2. Moderately scale-up best clone as determined in Specific Aim 1 to a working cell bank; characterize working cell bank and compare results to those obtained in Aim 1. Characterization will examine the identity, stability, and marker expression of the two clones. The significance of this work is its general applicability to hESC research and the benefit that will be reaped by the hESC research community once such a qualified reference standard is widely available.The end result and relevance of this project is a clone of human EC 2102Ep which closely represents the undifferentiated human ESC state. This clone will be produced under standardized conditions; it will be well-characterized, easy to use, and readily available. Availability of this clone is a necessary step to hasten the pace of sequential advances in hESC research. [unreadable] [unreadable] [unreadable]